BLID; a novel protein domain for interaction with the Bcl-2 family of proteins

ABSTRACT

In this invention, a novel protein interaction domain is provided along with several of its variants. This domain is involved in protein-protein interactions with the Bcl-2 family of proteins. It is named BLID (Bcl2 family of proteins Like Interaction Domain). Use of BLID and its variants for modulating cellular activity is presented. BLID, its variants and or anti-BLID antibodies could be useful as a screening tool as well as for discovery of drugs that help fight pathological states like degenerative diseases, cerebral or cardiac ischemic hypoxic disorders, cancer and autoimmunity.

REFERENCE TO RELATED APPLICATION

This application is related to and claims the benefit of U.S.Provisional Application Ser. No. 61/629,199 filed by applicant on Nov.14, 2011

A Sequence Listing is attached to this document.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates generally to the fields of molecular biology andmolecular medicine and more specifically to proteins involved in theregulation of cellular processes like apoptosis.

Apoptosis is one of the two main types of programmed cell death foundduring development in a wide spectrum of organisms from c. elegans tomammals. Apoptosis is a cellular process common to both physiologicaland pathological events in cells. Crucial for the execution of apoptosisare two families of proteins: caspases and the Bcl-2 family of proteins(Wyllie, 2010; Conradt, 2009)

The Bcl-2 family of proteins is comprised of two main functional groups:proapoptotic and antiapoptotic. Members of both subgroups interact witheach other in a complex network that controls the fate of the cell bytriggering or preventing apoptosis.

From the structural point of view, the antiapoptotic group of thisfamily is characterized by having four Bcl-2 Homology (BH) domains. Theyare called BH1, BH2, BH3 and BH4. The proapoptotic group is furthersubdivided on a multidomain group; which have BH1, BH2 and BH3 domainsand a BH3 only group with only one domain (BH3). The BH3-only group isfurther functionally subdivided in activators and derepressors,depending on their interactions with either proapoptotic multidomainproteins or with antiapoptotic-proapoptotic protein complexes (Wyllie,2010; Conradt, 2009).

Representative members of the antiapoptotic group in the Bcl-2 family ofproteins are: Bcl-2, Bcl-XL, Mcl-1, Bcl-W, Bfl-1, and Bcl-B. Members ofthe proapoptotic group of the Bcl-2 family are further subdivided intotwo groups: Bax, Bak, and Bok (multidomain group) and Bid, Bim, Bad,Puma, Noxa, and others (BH3-only group). These proteins interact witheach other in protein-protein interactions mainly through BH domains(Wyllie, 2010; Conradt, 2009).

An imbalance in apoptosis modulation can lead, via either excessive ordeficient activity, to pathogenic states like neurodegeneration, heartdisease, autoimmunity or cancer respectively (Nemec and Khaled, 2008;Tischner, 2010; Drag and Salvesen, 2010; Volbracht, 2001). Because theBcl-2 family of proteins plays such an important role in apoptosis itsmembers have been the targets of several approaches of drug discoveryefforts. These approaches include small molecule inhibitors, antisense(AS) oligonucleotides, ribozymes, etc. Among the members of the Bcl-2family currently under investigation are: Bcl-2, Bcl-W, Bcl-XL and Mcl-1(Ashkenazi and Herbst, 2008; Sasi, 2009).

A salient feature of apoptosis regulation is the redundant role of Bcl-2family members in these complex networks (Nemec and Khaled, 2008; Sasi,2009). Finding out how these redundancies occur has been a focus ofresearch as it has a direct impact on therapeutic efforts. One keyelement of this line of research is identifying new partners and theirnovel ways of interaction within this complex regulatory network.

We have identified a novel domain involved in apoptosis modulation viaits interaction with the Bcl-2 family of proteins. This novel domain wasidentified in lens epithelium-derived growth factor (LEDGF). BLID andantibodies against it can be used as a screening tool for characterizingthe presence and interactions of members the Bcl-2 family of proteins incells.

We think that several molecules that use this novel domain as a templatecan be also very useful in drug discovery efforts aimed at fightingdisease states like degenerative diseases, cerebral or cardiacischemic/hypoxic disorders, cancer and autoimmunity.

SUMMARY OF THE INVENTION

In accordance with the present invention, there are provided novel Bcl2family of proteins Like Interaction Domain (BLID) and several derivativemolecules thereof. In addition to these polypeptides, nucleic acidmolecules encoding BLIDs, vectors containing these nucleic acidmolecules and host cells containing such vectors are indicated. Theinvention also indicates antibodies that can specifically bind toinvention BLIDs. BLID, its derivatives and or anti-BLID antibodies couldbe useful for screening purposes, for instance in immunoassays aimed atcharacterizing the presence of BLID itself or Bcl-2 family members incellular samples.

A very important application of this invention is the use of BLIDcontaining polypeptides and its derivatives in the discovery of drugsthat help fight pathological states like cancer, autoimmunity,degenerative diseases, allograph rejection and infection.

The present invention indicates isolation procedures to identifyinteraction partners for BLID in mammalian cells. Identification ofnatural BLID partners can be very useful in the study of cell processeslike apoptosis. They can be also useful for the design of drugs aimed atmodulating programmed cell death in mammalian cells.

The present invention also indicates screening assays useful foridentifying agents, which can effectively alter the association of aninvention BLID with itself or with other proteins. By altering theself-association of BLID or by altering its interactions with otherproteins, an effective agent may increase or decrease BLID action andtherefore modulate cellular pathways that effect cellular processes likeapoptosis.

The invention also provides methods of altering the activity of BLID ina cell; wherein such increased or decreased activity of BLID canmodulate cellular pathways that effect apoptosis. For example, theactivity of BLID in a cell can be increased by introducing into the cella nucleic acid sequence encoding BLID or BLID derivatives and expressingit. Alternatively, BLID and BLID derivatives can be produced viarecombinant techniques or chemical synthesis and added to cells to beinternalized by the cells. BLID and BLID derivatives can also bemicroinjected into cells or otherwise introduced into cells in order tomodulate cell processes like apoptosis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Definition of the Bcl2 family of proteins Like InteractionDomain on LEDGF. The BLID domain (Bcl-2 family of proteins LikeInteraction Domain). LEDGF splice variants A) p75 NM_(—)033222(NP_(—)150091.2); B) p52 NM_(—)021144 (NP_(—)066967.3). Regionboundaries are indicated. PWWP: PWWP domain; CR1-5: Conserved ChargedRegions; NLS: Nuclear Localization Signal; A/T: A/T Hooks; IBD:Integrase Binding Domain. The position of BLID on LEDGF is labeled by asquare. C) BLID aminoacid sequence, variants 1 (SEQ ID NO: 1), 2 (SEQ IDNO: 2) and 3 (SEQ ID NO: 6), are shown. Residue positions are indicated.

FIG. 2. BLID secondary structure prediction. Top panel: Position ofα-helices (cylinders) from the structures of N1L (PDB 2UXE) and Bcl-XL(PDB 1MAZ) are shown (helices 2-5). Bottom panel: Secondary structurepredictions for BLID based on two different prediction algorithms, darkgray: Garnier-Robson algorithm (top sequence) and light gray:Chou-Fasman algorithm (bottom sequence). A, B and C represent BLIDvariants 1 (SEQ ID NO: 1), 2 (SEQ ID NO: 2) and 3 (SEQ ID NO: 6)respectively. Residue positions for N1L, Bcl-XL and LEDGF are indicated.

FIG. 3. Sequence alignment between Bcl-XL and BLID. Sequence alignmentbetween a partial BLID SEQ ID NO: 6 as target and Bcl-XL (PDB 1MAZ) astemplate in a threading analysis. Aminoacid similarities are indicatedas follows: (|): Identical residues, (:): very similar residues (PAMexchange matrix score: 1), (.): weakly similar residues (PAM exchangematrix score: −1). A bar indicates the BH3 motif in Bcl-XL. Criticalresidues for the BH3 domain are indicated. Residues are color-coded:hydrophobic residues: very light gray; acidic residues: light gray;basic residues: dark gray. Residue positions for Bcl-XL and BLID areindicated.

FIG. 4. Threading analysis of BLID. A Bcl-XL complex with Bim-BH3peptide (PDB 1PQ1) and Bcl-W (PDB 1O0L) were used as templates and BLIDSEQ ID NO: 06 was used as a target. A) Frontal view of a provisional 3Dstructure shows BLID helices in relationship with the Bim BH3 peptide.B) Side view of A. Threading energy was −2.4. Residues forming the BH3motif on Bim and proposed interacting residues on BLID have theirresidue position labeled and their contact surfaces (Van der Waalsradius) are represented by dots. On Panel A individual helices can beidentified, from right to left, helix 2 coming down on a 45 degreeangle, followed by helix 3 and finally helix 4 coming up in a 45 degreeangle, the helix corresponding to Bim-BH3 is seen in an horizontalorientation.

FIG. 5. Assaying phenotypic effects on mammalian cells of BLID and itsderivatives. Building blocks for molecules used in testing modulationeffect of BLID on cell processes like apoptosis. A) BLID B) BLIDderivatives: BLID linked to a Cell Penetrating Peptide molecule, shorterversions of BLID, BLIDIns (BLID with a small aminoacidic insertion),BLID-HM (BLID with mutations on some modeled helices); PolyX, aheterologous polypeptide linked to BLID, for example GST(Glutathione-S-Transferase); PolyY, another heterologous polypeptidelinked to BLID for example EGFP (Enhanced Green Fluorescent Protein).Building blocks can also be alternatively engineered in N or C terminalpositions to the original design. C) The molecules are either cloned onexpression vectors transfected and expressed on mammalian cells (left)or expressed on heterologous systems, purified and then added (right) tomammalian cells plated on 16, 96, etc well plates. D) Plates areanalyzed for phenotypic changes.

FIG. 6. Another example of a BLID model. BLID SEQ ID NO: 2 was used as atarget and the crystal structure of Bcl-XL (PDB 1PQ0) as template in athreading analysis. Threading energy was −3.8. A) Frontal view withmodeled helices for BLID. B) Side view of A. Proposed interactingresidues on BLID have their residue position labeled and their contactsurfaces (Van der Waals radius) are represented by dots. On Panel Aindividual helices can be identified, from right to left, helix 2 is thefirst one coming down and out of the figure, to the left helix 3 is atthe bottom and helix 4 is on top.

FIG. 7. Comparison of two BLID models obtained via homology modelling. Athreading analysis was done using BLID SEQ ID NO: 2 as a target and twodifferent crystal structures of anti-apoptotic proteins in complexeswith their corresponding BH3 only pro-apoptotic molecules as templates.The experimental structures are Bcl-XL in complex with a BH3 peptidefrom Bim (PDB 1PQ1) and CED-9 in complex with a BH3 peptide from EGL-1(PDB 1TY4). The threading energy was −3.6 and −2.5 respectively. Ribbonsindicate structural features; dots indicate contact surfaces (Van derWaals radius). White is used for anti-apoptotic molecules, dark gray forpro-apoptotic molecules and light gray for BLID. Panels, from left toright, represent clockwise rotation of the models.

FIG. 8. Examples of BLID protein and nucleotide sequences. A) LEDGFisoform p52 derived sequences. B) LEDGF isoform p75 derived sequences.Examples of BLID polypeptide sequences are indicated: 1 (SEQ ID NO: 1);2 (SEQ ID NO: 2); 3 (SEQ ID NO: 6); 4 (SEQ ID NO: 12); 5 (SEQ ID NO:13); 6 (SEQ ID NO: 19). An arrow indicates N-terminal portion ofindividual polypeptides. Nucleotide sequences are also indicated.

DETAILED DESCRIPTION OF THE INVENTION

The Bcl-2 family of proteins is comprised of two main functional groups:proapoptotic and antiapoptotic. Members of both subgroups interact witheach other in a complex network that controls the fate of the cell bytriggering or preventing apoptosis.

From the structural point of view, the antiapoptotic group of thisfamily is characterized by having four Bcl-2 Homology (BH) domains. Theyare called BH1, BH2, BH3 and BH4. The proapoptotic group is furthersubdivided into a multidomain group; which has BH1, BH2 and BH3 and aBH3 only group with only one domain (BH3). The BH3-only group is furtherfunctionally subdivided in activators and de-repressors depending ontheir interactions with either proapoptotic multidomain proteins or withantiapoptotic-proapoptotic protein complexes (Drag and Salvesen, 2010;Conradt, 2009; Wyllie, 2010)

Representative members of the antiapoptotic group in the Bcl-2 family ofproteins are: Bcl-2, Bcl-XL, Mcl-1, Bcl-W, Bfl-1, and Bcl-B. Members ofthe proapoptotic group of the Bcl-2 family are further subdivided intotwo groups: Bax, Bak, and Bok (multidomain group) and Bid, Bim, Bad,Puma, Noxa, and others (BH3-only group). These proteins interact witheach other in protein-protein interactions mainly through BH domains.(Drag and Salvesen, 2010; Conradt, 2009; Wyllie, 2010)

An imbalance in apoptosis modulation can lead, via either excessive ordeficient activity to pathogenic states like neurodegeneration, heartdisease, autoimmunity or cancer respectively (Nemec and Khaled, 2008;Tischner, 2010; Drag and Salvesen, 2010; Volbracht, 2001). Because theBcl-2 family of proteins plays such an important role in apoptosis itsmembers have been the targets of several approaches of drug discoveryefforts. These approaches include using peptides, small moleculeinhibitors, antisense (AS) oligonucleotides, ribozymes, etc. Among themembers of the Bcl-2 family currently been investigated are: Bcl-2,Bcl-W, Bcl-XL and Mcl-1 (Ashkenazi and Herbst, 2008; Sasi, 2009;Leibowitz and Yu, 2010).

One of the fundamental traits of cancer cells is their resistance toapoptosis. As a result a great deal of research has been devoted toovercome this resistance and use the pre-existing apoptotic machineryagainst tumor cells (Sasi, 2009; Leibowitz and Yu, 2010).

The more advanced drug against a Bcl-2 protein family member isOblimersen (an antisense agent against Bcl-2) currently in clinicaltrials phase III. The success of this drug has been uneven with positiveresults in some tumors types like chronic lymphocytic leukemia andmelanoma and disappointing results in others like prostate, myeloma, andacute myeloid leukemia. (Ashkenazi and Herbst, 2008). Another antisensedrug is AS Bcl-2 (G3139) (Sasi, 2009).

Another approach to drug discovery is based on small molecules acting asinhibitors of anti-apoptotic members of the Bcl-2 family of proteins.These compounds are commonly known as BH3 mimetics. An example of thesecompounds is ABT-263, a small molecule that binds in the subnanomolarrange to Bcl-2, Bcl-XL, and Bcl-W. GX-15-070 (obatoclax) is anotherinhibitor of 5 members of the Bcl-2 family of proteins, which is inphase II of clinical trials. In addition, antagonists of Mcl-1 are beingdeveloped (Ashkenazi and Herbst, 2008). Other examples are ABT-737 (aBcl-2 XI antagonist) and WL-276. Another group of small molecule Bcl-2inhibitors are (−)-Gossypol (AT-101) and a less toxic derivative labeledapogossypol (NSC736630) (Sasi, 2009; Akiyama, 2009). AT-101 is currentlyin phase I of clinical trials (Ashkenazi and Herbst, 2008).

At the post-transcriptional level Bcl-2 has been targeted with ribozymes(Sasi, 2009). Bim has been the target in studies using siRNA in thecontext of fighting sepsis in a murine model (Hattori, 2010).

It is also important to note that modulation over Bcl-2 can affectprogrammed necrosis also and as such be a promising candidate forclinical programmed necrotic cancer therapy (Sasi, 2009).

After filing our initial application we found out that a proteinreported as BRCC2 (breast cancer cell 2) (Kasid 2003; Broustas 2004;Lomonosova 2008) had its name changed to BLID (BH3-like motifcontaining, cell death inducer) (Broustas 2010). This protein is relatedto Bad, Puma etc both functionally (proapoptotic) and structurally(single BH3 motif); but it has a variation on its BH3 motif (it has noaspartate) and therefore is classified as BH3-Like. It should be notedthat the name we used stands for a different acronym (Bcl2 family ofproteins Like Interaction Domain) it is derived from a different protein(LEDGF) and it is a domain.

PC4- and SF2-interacting protein 1 (Psip1) is encoded by the PSIP1 gene.This gene encodes two isoforms, p75 (530 aa) and p52 (333 aa). These twoproteins share the first 325 N-terminal residues. The most commonly usedname for this protein is lens epithelium-derived growth factor (LEDGF),it is also known as dense fine speckles 70 kDa autoantigen or DSF70(Sutherland, 2006; Hendrix, 2010; Sugiura, 2007).

LEDGF was initially isolated and characterized as a transcriptionalco-activator (Ge, 1998). An independent group determined its role as asurvival factor in lens epithelial cells under different environmentalstresses (Singh, 1999). LEDGF has been proposed as a transcriptionalregulator of several stress-related genes (Matsui, 2001). Theantiapoptotic effect of LEDGF is believed to occur via transcriptionalactivation of stress related genes (Sutherland, 2006).

This protein is also an autoantigen found in patients with atopicdermatitis and other inflammatory disorders involving an imbalance ofapoptosis regulation. LEDGF is also cleaved by caspases during apoptosis(Sutherland, 2006). The p75 isoform of LEDGF co-precipitates withintegrase (IN) from human immunodeficiency virus type 1 (HIV-1). Thisdiscovery brought a lot of attention to this protein; suggesting a rolein integration of the HIV (Cherepanov, 2003). In addition LEDGF has beenconnected with oncogenesis (Hendrix, 2010). This protein has beencovered in several US patents (Shinohara, et al U.S. Pat. No. 6,750,052;Debyser, et al U.S. Pat. No. 7,514,233 and U.S. Pat. No. 8,008,470;Goldstein, et al. U.S. Pat. No. 8,168,393)

Several domains have been identified in LEDGF. The N-terminal PWWPdomain, (residues 1-93) belongs to the family of Tudor domains involvedin chromatin binding (Hendrix, 2010; Shun, 2008). The PWWP domainincludes a Pro-Trp-Trp-Pro motif, and it has been identified in about 60eukaryotic proteins. The next domain is CR1 (residues 94-142); one ofseveral conserved charged regions (CR) found in LEDGF. These regionscontain a high concentration of positively charged residues and arethought to be involved in electrostatics interactions with DNA chromatin(Hendrix, 2010; Botbol, 2008). A nuclear localization domain (NLS) isfound between residues 146-156. This domain is mainly connected to thenuclear localization of this protein as well as contributing tochromatin binding (Meehan, 2009; Botbol, 2008). A A-T Hooks domain(residues 178-198) contains two motifs, that along with the NLS form atripartite element that cooperates with the PWWP domain in chromatinbinding (Garcia-Rivera, 2010; Hendrix, 2010; Botbol, 2008).

Regions CR2 (residues 199-266), CR3 (residues 267-325) and CR4 (residues326-339) have similar properties to CR1. These are thought to beinvolved in nonspecific electrostatic interactions with chromatin DNA.Segments of CR2 have high contents of lysines, which are proposed to betargets for posttranslational modifications like SUMOylation,ubiquitination, and glycosylation. (Garcia-Rivera, 2010; Meehan, 2009;Hendrix, 2010). A stretch of serines (S271, S273 and S275) in CR3 hasbeen identified as having phosphorylated Ser/Thr sites and it has beenproposed to be a target for protein kinase casein kinase 2 (PKCK2)(Garcia-Rivera, 2010). CR2 and CR3, with no autonomous chromatin bindingactivity appear to enhance the activity of the N-terminal domainsspecifically involved in chromatin binding. (Llano, 2006; Meehan, 2009).

The Integrase Binding Domain (IBD) (residues, 347-429) (Meehan, 2009;Hendrix, 2010; Shun, 2008; Botbol, 2008) is involved in protein-proteininteractions with the Integrase (IN) from human immunodeficiency virustype 1 (HIV-1). The region CR5 (residues 443-530) located C-terminalfrom IBD, does not have a high concentration of charge residues but itis conserved. CR5 also contains four demonstrated Ser/Thr phosphosites,three of them clustered near the C-terminal end (Garcia-Rivera, 2010).

The term “functional equivalent”, when used herein as a modifier ofinvention BLID, or polypeptide fragment thereof, refers to a polypeptidethat exhibits functional characteristics similar to a BLID. For example,one biological activity or function of BLID is the ability to bind,preferably in vivo but also in vitro, to a member of the Bcl-2 family ofproteins, like Bid or Noxa.

Preferably, a “functional equivalent” may be a polypeptide and itsencoding nucleic acid that displays substantially similar activitycompared with BLID or fragments thereof in a suitable assay for themeasurement of biological activity or function. For instance afunctional equivalent could display between 20-40%, 40-50%, 60-70%,70-80%, 80-90% or even more than 100% activity in comparison with BLIDin cell survival assays.

Also a “functional equivalent” may be a polypeptide able to function ina similar fashion; both in vivo or in vitro; when compared with theinvention BLID and fragments thereof. In an in vitro example, a peptidederived from a BLID sequence is used either in its original compositionor further chemically modified (for instance with the substitution of aresidue with an amino acid derivatives like 3,4-dihydroxy-phenylalanine,cyclohexyl-glycine, etc or alternative amino acids like D-Ala). Thisfunctional equivalent peptide is used to disrupt the binding between anantibody raised against BLID and fragments thereof in an immuno assay,like an ELISA. The reduced binding activity will diminish by at least10%, more preferably between about 10% and 35%, even more preferablybetween about 35% and 45%, and most preferably between about 45% and50%.

When referring to a polypeptide which exhibits “significant structuralhomology” to polypeptides described in this invention (BLID) we meanpolypeptides that while having low sequence identity as compared to theBLID polypeptides, are predicted to be related molecules by virtue ofsharing significant structural homology with the BLID polypeptidesequences. The structural homology can be calculated using differentstructural genomics computational methods for structure analysis, usingcomparison between experimental structures and or models. Asexperimental BLID structures become available other software likeProbability of Identity 2 (PRIDE2) (Vlahovicek et al 2005) can be used.For instance it can be considered that a polypeptide has a significantstructural homology to BLID if it has a PRIDE2 score between 0.6 and0.8; preferably a PRIDE2 score >0.85 and even more preferably a PRIDE2score >0.9.

We have identified the BLID domain to have a similar topology to aregion of Bcl-XL and Bcl-W and CED-9, three members of the Bcl-2 familyof proteins. Bcl-2 family of proteins generally are known to interactwith several members of both proapoptotic and antiapoptotic members ofsaid family of proteins, such as Bcl-2, Bcl-XL, Mcl-1, Bcl-W, Bfl-1,Bcl-B, Bax, Bak, Bok, Bid, Bim, Bad, Puma, Noxa, and others.

We hypothesized that, despite a low sequence similarity between aportion of LEDGF and the Bcl-2 family of protein members, they shared acommon overall fold. Conservation of protein structure has been found tobe stronger than protein sequence during evolution (Graham et al 2008).Recent discoveries of viral proteins with very little sequence identity(less than 15%), but a similar protein fold and function to members ofthe Bcl-2 family of proteins illustrates this point (Cooray, 2007;Aoyagi, 2007; Kvansakul, 2008 and Douglas, 2007). Such common fold wouldin turn suggest a similar function and therefore indicate the presenceof a novel domain in LEDGF. The presence of such domain would havedirect implications on the biological role of LEDGF as well asconstituting a new avenue for modulation of cellular processes such asapoptosis.

The divergence of residue types observed between N1L and Bcl-XL in theinteraction with a Bim-BH3 peptide indicates the flexibility ofarrangement possible for the same type of biologically meaningfulinteraction, for instance the presence of charged residues in N1L (D35;R71) instead of hydrophobic ones in Bcl-XL (Y101; A142) (Cooray 2007).This flexibility is also observed for BLID, suggesting a possible rolein similar interactions with a similar peptide motif from a member ofthe Bcl-2 family of proteins.

Finding similar results in homology modelling exercises using threemembers of the Bcl-2 family of proteins (Bcl-XL, Bcl-w and the Bcl-2 c.elegans homologue CED-9) and BLID strengthens the case for the presenceof a partial structural homolog in this newly defined LEDGF domain.

It is important to note that BLID fold similarities have been calculatedwith anti-apoptotic members of the Bcl-2 family of proteins, whichsuggests a similar activity that is an anti-apoptotic activity, forBLID. This anti-apoptotic role for BLID suggests that the more likelyinteraction partners for this polypeptide are the pro-apoptotic membersof the Bcl-2 family of proteins (like Bim, Bid, Noxa, Puma, Bax etc) orwith yet unknown proteins with a similar pro-apoptotic role.

All of the above analysis suggests that there is a domain in LEDGF witha similar fold to members of the Bcl-2 family of proteins. This domaincan be involved in a new type of protein-protein interaction withmembers of the Bcl-2 family of proteins. This constitutes a novelinteraction for LEDGF, which previously has been regarded as atranscriptional co-activator.

Because of the above mentioned potential novel interactions, BLID andits derivatives can be used as a way of modulating cell physiology;mainly apoptosis but also autophagy and necrosis. Molecules that disruptthe interactions between BLID, its derivatives and their cellularinteracting partners can also be used for therapeutic or diagnosticmeans. As a result, this invention can open new avenues in the fightagainst disease states like degenerative diseases, stroke, autoimmunityand cancer.

Polypeptides containing BLID and its variations can be expressed andpurified using known recombinant techniques (Sambrook, 1989). These BLIDcontaining polypeptides can also be fused to other polypeptides toenhance solubility, provide structural stability, direct cellularlocalization, facilitate uptake by the cell, facilitate purification,act as a tag for detection etc. Among these fused polypeptides (but notlimited to) are GST (Glutathione-S-Transferase), MBP (Maltose BindingProtein), His-Tag, an antibody, HA-Tag, EGFP, Inteins, Streptavidin, TAT(HIV derived cell penetrating peptide) etc.

Nucleotide sequences are also provided which code for the polypeptidesdefined for BLID and its variants, SEQ ID NO: 26 and SEQ ID NO: 27 codefor the longest variants (variants 1 (SEQ ID NO: 1) and 4 (SEQ ID NO:12) respectively. The nucleotide sequence for the shorter sequences canbe assigned from the corresponding polypeptide sequences already definedand from FIG. 8. These nucleotide sequences are used for designingprimers for PCR as well as guides for other recombinant technologyprocedures like cloning, hybridizations, expression etc. Thesenucleotide sequences are also useful as templates to design probes usedfor isolating other similar sequences from genetic libraries.

BLID containing molecules can be used to isolate interacting partnersusing common assays for studying protein-protein interactions.Non-limiting examples of these are: protein complexes isolation viaaffinity chromatography, affinity tags (like GST or MBP) orImmunoprecipitation. Biochemical methods for protein complexespurification like FLAG-tagged affinity purification or tandem affinityprocedure (TAP) can also be employed to isolate BLID partners. Theresulting enriched protein fractions are subjected to IsoelectricFocusing Electrophoresis (IEF) and or SDS-PAGE Electrophoresis. Twodimensional-differential gel electrophoresis (2D-DIGE) can also beemployed. Specific bands or spots are then analyzed by matrix assistedlaser desorption ionization time of flight mass spectrometry (MALDI-TOFMS). Afterwards, the obtained sequences are compared againstnon-redundant protein databases like NCBI non-redundant protein sequencedatabase. Additional techniques like phage display and proteinmicroarray technology can also be useful to identify BLID partners.Genetic based strategies like yeast two hybrid and its mammaliancounterparts can be used to isolate BLID partners as well.

Once BLID containing molecules interaction partners have been isolated,a number of drug discovery efforts can be designed around the BLIDcontaining molecules—partner interactions. Efforts would be directed toeither disrupt or promote said interactions. Non-limiting examples ofthese drug discovery efforts are the use of combinatorial chemicallibraries, combinatorial peptide libraries, antisense and InterferingRNA (siRNA, shRNA etc) techniques and structure-based computer screeningfor binding sites similarities.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLES Example 1 Definition of the Bcl2 Family of Proteins LikeInteraction Domain on LEDGF

We called this domain BLID (Bcl2 family of proteins Like InteractionDomain). The overall position of BLID within LEDGF and the aminoacidicsequence of three BLID variants modelled after LEDGF/p52 are shown inFIG. 1. BLID variants 4 (SEQ ID NO: 12), 5 (SEQ ID NO: 13) and 6 (SEQ IDNO: 19) are modelled after LEDGF/p75; they have the same N-terminalposition as variants 1 (SEQ ID NO: 1), 2 (SEQ ID NO: 2) and 3 (SEQ IDNO: 6) respectively but differ in their last 8 C-terminal residues. AllBLID sequences are shown in the sequence list.

We analyzed BLID for secondary structure. Two different secondaryprediction algorithms were applied: Chou-Fasman and Garnier-Robson,using the program Protean 3.02 (DNAStar software suite). We also useddata from the crystal structures of a member of the Bcl-2 family ofproteins, Bcl-XL and N1L. N1L is a Vaccinia virus protein, which sharesfold and function with Bcl-XL and other members of the Bcl-2 family ofproteins despite a very low sequence identity (Cooray, 2007; Aoyagi,2007).

FIG. 2 shows the resulting secondary structure predictions mapped ontothe primary sequence of LEDGF and compared with known structuralelements found in NIL and Bcl-XL. The analysis predicts a similar arrayof multiple helices for BLID when compared with Bcl-XL and N1L. Some ofthe predicted helices for BLID are in similar positions to helices foundin N1L and Bcl-XL.

FIG. 3 shows the results of an alignment between Bcl-XL and BLID variant3 from a homology modelling test (manual mode) done with Swiss-PdbViewer4.01 (OS X). The target sequence used was BLID variant 3 and thetemplate used was the crystal structure of Bcl-XL (pdb code 1MAZ). Theresulting provisional 3D structure shows several residues correspondingto helices 2-4 of Bcl-XL are found with several degrees of conservationin BLID. The threading energy calculated for this arrangement was −1.2.

An arrangement of residues similar to the BH3 motif in Bcl-XL is foundin a segment of BLID (F293-L302). This segment is located near the BH3region of Bcl-XL. In addition, this region contains a cluster ofidentical residues between the two polypeptides. This data suggests thepresence of a similar BH3 motif in BLID.

BH3 domains have been difficult to identify from sequence alone becausethe pattern of residues is poorly conserved and there are no invariantresidues. In order to identify these domains a combination of sequenceand structural analyses, as well as a common molecular mechanism forbinding to other Bcl-2 family of proteins may be necessary (Sinha 2008).

FIG. 4 shows the basic structural elements of BLID. These elements arerepresented on a provisional 3D structure obtained in a manual modehomology modelling test done with Swiss-PdbViewer 4.01 (OS X). Astructure of Bcl-XL in complex with a peptide containing the BH3 motifof Bim (PDB 1PQ1) was superimposed with a Bcl-w (PDB 1O0L) structure andthen both were used as template with BLID SEQ ID NO: 6 primary sequenceas target in a threading analysis. The threading energy calculated forthis arrangement was −2.4.

Several residues in BLID are in position to accommodate the amphipathicnature of the incoming BH3-peptide from Bim. In a front view (panel A)several hydrophobic residues are found in BLID proposed helices 2 and 3and in the loops among helices 2-4. These hydrophobic residues arefacing the hydrophobic face of the BH3 helix. It appears as if the BH3peptide is in the process of advancing in the frame created by BLID'sthree proposed helices.

As shown in Panel A, F19 and A22 from BLID are involved in the initialinteraction with L94 and F101 from Bim-BH3. Further positioning of theBH3 peptide into BLID's frame (moving to the left on Panel A) willengage a third hydrophobic residue (L90) with the hydrophobic part ofBLID's frame.

BLID residues are distributed in two distinct sections along its frame.The first is a hydrophobic sector composed of F19, A22, M27, L28, A36and A37, which stretches from the C-terminal portion of the proposedhelix 2 to the loop between helix 3 and 4. The second section is acharged sector composed of E44, E48 and H51 on helix 4. These twosections are position facing the hydrophobic and charged faces of theincoming BH3 peptide as shown in a side view (panel B). If the BH3peptide is moved further to the left in Panel A, it will come intocontact with more matching elements of BLID's frame in a similar fashionas the interaction described with other Bcl-2 family of protein members.

The divergence of residue types observed between N1L and Bcl-XL in theinteraction with a Bim-BH3 peptide indicates the flexibility ofarrangement possible for the same type of biologically meaningfulinteraction, for instance the presence of charged residues in N1L (D35;R71) instead of hydrophobic ones in Bcl-XL (Y101; A142) (Cooray 2007).This flexibility is also observed for BLID, suggesting a possible rolein similar interactions with a similar peptide motif to a member of theBcl-2 family of proteins. Additional threading analysis of BLID variantssuggests variants 1-3 are more effective in adopting the proposed fold.

Another model for BLID is shown in FIG. 6. In this example a threadinganalysis was done using a murine Bcl-XL (PDB 1PQ0) as template and BLIDSEQ ID NO: 2 as target. A favorable energy threading of −3.8 wasobtained, again in line with the ones mentioned above. In this analysismore of the proposed interacting residues in the BLID frame are locatedon helices. A different overall arrangement is presented with a narrowspace created by helices 3 and 4 in an antiparallel conformation, withthe C-terminal of helix 2 making a re-enforcement of the entrance to thenarrow canal made by helices 3 and 4 (Panel A).

A side view (Panel B) reveals the distribution of residues on the BLIDframe with the hydrophobic residues at the bottom and the charged onesat the top of the channel created by the three helixes. The hydrophobicand charged sections of BLID are arranged to accommodate the amphipathicnature of a BH3 containing peptide like the one found in Bim. Inaddition a side view allows to see several residues from the hydrophobicsection of BLID in very good alignment, they are: F19 and A22 (helix 2),M27 (just before helix 3) and A36 and A37 (loop between helix 3 and 4).In. very good alignment are also the following residues from the chargesection: E44, E48 and H51, all of them on helix 4.

FIG. 7 shows A threading analysis was done using BLID SEQ ID NO: 2 as atarget and two different crystal structures of anti-apoptotic proteinsin complexes with their corresponding BH3 only pro-apoptotic moleculesas templates. The experimental structures are Bcl-XL in complex with aBH3 peptide from Bim (PDB 1PQ1) and CED-9 in complex with a BH3 peptidefrom EGL-1 (PDB 1TY4). The threading energy was −3.6 and −2.5respectively.

The finding of similar results in homology modelling exercises usingthree members of the Bcl-2 family of proteins (Bcl-XL, Bcl-w and CED9, ac. elegans Bcl-2 homologue) and BLID strengthens the case for thepresence of a structural homolog in this newly defined LEDGF domain.

All of the above analysis suggests that there is a domain in LEDGF witha similar fold to members of the Bcl-2 family of proteins. This domaincan be involved in a new type of protein-protein interaction withmembers of the Bcl-2 family of proteins. This constitutes a novelinteraction for LEDGF, which previously has been regarded mainly as atranscriptional co-activator.

Because of the above mentioned potential novel interactions, BLID andits derivatives can be used as a way of modulating cell physiology;mainly apoptosis but also autophagy and necrosis. As a result, thisinvention can open new avenues in the fight against disease states likedegenerative diseases, stroke, autoimmunity and cancer.

Example 2 Modulating Apoptosis in Mammalian Cells Using BLID and itsDerivatives

BLID and several of its derivatives are realized. These molecules areassayed in two different ways to modulate apoptosis in mammalian cells.In the first way BLID and some of its derivatives are cloned intoexpression vectors like pcDNA1 (Invitrogen), etc and then transfectedinto mammalian cells (like CHO, Hela, Jurkat, HEK293 etc) and expressed.Stably transfected cell lines can also be established. Cells andcontrols are then challenged with apoptosis-inducing stimuli(actinomycin D, Staurosporine, etoposide etc) and phenotypic changesmonitored via microscopy and subsequent assays like western blot forcaspases, poly (ADP-ribose) polymerase (PARP) etc.

The second way uses already purified BLID and derivatives, adding themto mammalian cells instead of expressing them in the target cells. FIG.5 shows both approaches for assessing BLID. Different variants of BLIDand its derivatives (expressed on mammalian cells or as purifiedpolypeptides) are added to cells (with or without helpers likelipofectamine etc) or microinjected. In FIG. 5 several BLID andderivatives are realized. Cells are challenged with apoptosis-inducingagents, and then microscopy and subsequent assays are conducted todetermine phenotypic and other molecular changes.

In FIG. 5 BLID variants are sequences from SEQ ID NOs: 1 to 24.Derivatives are created by modifying BLID in different ways: a) usingshorter versions of BLID, for instance sequence 7, b) engineering shortaminoacidic insertion into BLID (BLIDIns variant), c) designingmutations into predicted helical regions (BLID-HM), d) heterologouspolypeptides cloned as fusion proteins to BLID or the above-mentionedvariants. Examples of these polypeptides are EGFP and GST, e) Dependingon delivery route, all of the above mentioned combinations could also becombined with Cell Penetrating Peptides (CPP). All of these buildingblocks can be also alternatively engineered in N or C terminal positionsto the original design

Cell Penetrating Peptides (CPP) are intended to help translocate BLID tothe cell from the culture medium. Examples of these are: TAT,penetratin, transportan and polyarginines and polylysines of differentlengths, typically around 9 residues long (Herce and Garcia, 2007).

Linkers used are aminoacids or chemical groups. Examples of amminoacidsused for linkers are Glycine and Serine, with variable length, forinstance between 3-15 residues. A preferred length would be between 3-5residues long. Chemical groups used as linkers can be for instancethioesters.

Depending on the particular composition of an engineered molecule it canbe produced entirely using recombinant technology, like in the exampleof a CPP-BLID molecule in which the CPP is cloned in frame C-terminal ofBLID with a Gly₃-Ser linker in between them. In another variant,building blocks are produced individually via recombinant technology orchemically synthesized and then chemically linked. Alternatively, awhole variant molecule is made via chemical synthesis.

Cell lines which are used in particular disease models, can also betested with BLID and its derivatives. Examples of those are N27 cells(mesencephalic dopaminergic neuronal cell line) used in Parkinson'sdisease studies (Carvour, 2008) and human retinal pigment epithelium(RPE) cells, which are used in Age-related macular degeneration (ARMD)studies (Jiang, 2005).

Example 3 In Vivo Assay for BLID Activity

HEK 293 or HeLa cells are seeded at ˜10⁶ cells per 15 cm dish. 72 hourslater cells are transfected with pcDNA3.1 or pCruzHA vectors containingone of the following constructs: BLID sequences SEQ ID NO: 2, SEQ ID NO:8, SEQ ID NO: 11 and SEQ ID NO: 17, BLID sequences SEQ ID NO: 2, SEQ IDNO: 8, SEQ ID NO: 11 and SEQ ID NO: 17 with a N-terminal fusion of GST,LEDGF/p52 and LEDGF/p75. Fugene 6 (Roche Diagnostics) or Lipofectamine2000 (GIBCO BRLTechnologies Inc.) are used for the transfections. Inaddition controls of mock transfection and individual plasmids are used.24-48 hours later apoptosis is induced by adding to the cellsstaurosporine (1 uM, or 250 nM) for 1.5, 3, 6, 12 and 24 h. Afterwards,cells are analyzed for apoptosis.

Microscopy: Cells are first analyzed by morphological changes, celldetachment, cells shrinkage etc using an inverted microscope (like anOlympus IX70 Microscope) equipped with Hoffman modulation contrast.Cells are counterstained with Hoechst 33342 and visualized directlyusing a 60× water immersion objective under an epifluorescence anOlympus BX50 (Scientific Instruments) microscope equipped with a digitalcamera system (digital SPOT camera system (Diagnostic Instruments)).Nuclei of cells that exhibited marked chromatin condensation,margination, or fragmentation are counted as apoptotic. Approximately200 nuclei distributed in >10 different fields are counted in at leastthree independent double-blind experiments. Cells are selected forcounting of apoptotic nuclei by their expression of BLID variants andBLID containing polypeptide using their corresponding tagged molecules;in the case of pCruzHA-based constructs, by an anti-HA antibody (ratmonoclonal horseradish peroxidase (HRP)-conjugated anti-HA antibody(Roche Diagnostics)

When pCruzHA plasmids are used for transfection, cells are seeded oncoverslips and fixed for 15 min at room temperature with 3.7%paraformaldehyde and permeabilized in PBS-0.2% Triton X-100 for 5 min.Coverslips are then incubated with rabbit anti-HA antibody for 2 h.Following three washes with PBS, cells are incubated with Alexa 488 goatanti-rabbit for 1 h, washed with PBS, mounted on glass slide withVectashield Mounting Medium containing 4′,6-diamidino-2-phenylindole,and examined under a fluorescence microscope. Localization of HA-taggedpolypeptides as well as nuclear condensation is determined.

Cellular caspase activity: Activity in transfected and untreated cellsis determined by cleavage of the fluorogenic substrate DEVD-AMC andexpressed in relative fluorescence units (RFU), from which the value ofthe untreated control is subtracted.

Cells are seeded in black, clear-bottomed 96-well plates (10⁴ cells perwell). After 1.5, 3, 6, 12 and 24 h of subjecting cells to apoptoticstimuli, cells are incubated with 50 μl of 3× caspase buffer [150 mMHepes pH 7.4, 450 mM sodium chloride, 150 mM potassium chloride, 30 mMmagnesium chloride, 1.2 mM ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 30%sucrose, 10% CHAPS, and 1.5% NP-40], 30 mM dithiothreitol (DTT), 3 mMphenylmethanesulphonylfluoride (PMSF), and 75 μM of the fluorogenicpeptide substrates Ac-DEVD-AMC (caspase-3/7) or Ac-VDVAD-AMC (caspase-2)for 2 h at 37° C., followed by incubation at room temperature for 12 h.TRAL/actinomycin D treatment is used as a control for caspase-3/7activation, whereas STS is used as a control for caspase-2 activation.Absorbance is then read in a Microplate Fluorescent Reader (like theFLX800 (Bio-tek Instruments)) at excitation of 360 nm and emission of460 nm. Fold activity is determined by normalizing to one the absorbancevalues for untreated cells.

Cell survival is determined using the standard3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)assay (Sigma-Aldrich). As an alternative, second-generation tetrazoliumderivatives (e.g.,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) or4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate (WST-1)) can be used.

Cells are seeded in 96-well plates (10⁴ cells per well), after 1.5, 3,6, 12 and 24 h of subjecting cells to apoptotic stimuli they are washedwith phosphate buffered saline (PBS), and fixed in 4% paraformaldehydefor 1 h at 4° C. Cells are then washed three times with distilled water,and Accustain Crystal Violet solution (Sigma-Aldrich) (1:4) is added toeach well followed by incubation for 20 minutes at room temperature.Plates are washed with distilled water to remove excess dye and thendried at room temperature. Acetic acid (10% v/v) is added to each wellfor 10 minutes and absorbance is measured at 570 nanometers (nm) using amicroplate reader (like the μQuant (Bio-tek Instruments)).

Cell viability can be also determined using a modified(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MU) assay(Sigma-Aldrich, St. Louis, Mo.). Briefly, cells are seeded in 96-wellplates (10⁴ cells per well) and then after 1.5, 3, 6, 12 and 24 h ofsubjecting the cells to apoptotic stimuli MU is added to each well(final concentration, 1 mg/ml) and plates are incubated in a 5% CO2incubator at 37° C. for 1 h. Plates are centrifuged at 2,000 rpm for 30minutes. Supernatants are discarded and 150 μl of dimethyl sulfoxide(DMSO), are added to each well. Absorbance is measured at 450 nm.

Experimental alternatives to this example can be found below:

Other alternative cells to be used are: Jurkat, Normal human epidermalkeratinocytes (NHEK), HepG2, HCT116, PC3 and mouse LensEpithelium Cells(LEC). Alternative methods of inducing apoptosis are subjecting thecells to actinomycin D, cisplatin (50 uM), or oligomycine (5 uM) pluscarbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (1 uM),anti-CD-95 (4 ug ml) plus CHX (cyclohexamide) (2 ug/ml), exposing cellsto 100 to 500 J/m² UV radiation, 200 mJ/cm² UV-C and 100 uM etoposide.

In this example the overexpression of BLID reduces or abolishes theapoptotic effect produced by the apoptotic stimuli therefore acting asan anti-apoptotic molecule and promoting cell survival. This effect ofBLID constitutes one of the main avenues for drug development based onthis novel protein-protein interaction domain.

Example 4 BLID Polypeptide Purification

BLID SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are amplified by PCRfrom a plasmid derived from plasmid GST-K-p52 (Ge, 1998) using primersdesigned using the DNA SEQ ID NO: 26. Additionally oligonucleotidesencoding for a CPP (TAT-domain) are ligated to the amplified PCRfragments. The resulting sequences encode for a TAT-BLID fusedpolypeptide. Amplified PCR fragments are sub-cloned into cloning vectors(pBluescritp KS+ (Stratagene)), and verified by sequencing, Theconstructs are then cloned into pGEX plasmid (pGEX, Amersham, N.J.).

Clones are verified by sequencing. Vectors are transformed into E. coliand expressed. For protein expression E. coli Rosetta (pLysS) cells(Novagen, Madison, Wis.) are used, these are BL21 derivative designatedto enhance the expression of eukaryotic proteins containing codonsrarely used in E. coli.

Starter cultures of 5 ml Luria-Bertani (LB) medium containing 100 mg/mlampicillin are inoculated, with a BL21 recombinant clone. The culturesare grown overnight at 250 rpm and 37° C. One milliliter of theovernight culture is added to 100 ml LB medium supplemented with 100mg/ml ampicillin and further incubated at 37° C. up to an OD600 of 0.5.Culture is induced with IPTG at concentrations of 0.5 and 1 mM at anOD600 of 0.5, and at temperatures of 25° C. and 37° C. until reaching anOD of 2 at 600 nm.

Cells from induced and un-induced cultures are harvested bycentrifugation (4000 g, 25 min, 4° C.) followed by two washing stepswith buffer A (10 mM Na2HPO4, 2 mM KH2PO4, 150 mM NaCl, 2.5 mM KCl, pH7.5) at 4000 g for 25 min, and stored at −80° C. until use. Proteinextraction is performed by resuspending the cell pellet in one-fifth ofthe original culture volume of buffer B (10 mM Na2HPO4, 2 mM KH2PO4, 150mM NaCl, 2.5 mM KCl, pH 7.5 and 1% Triton X-100). The cells aredisrupted by sonication (5 20-sec bursts). The supernatant is collectedby centrifugation at 4° C. for 30 min at 13 000 rpm. Both pellets andsupernatants are stored at 4° C.

The supernatant containing the soluble GST-BLID and GST-TAT-BLIDrecombinant proteins are loaded on a GSTrap FF affinity column (1 ml;Amersham Biosciences) pre-equilibrated with buffer A (10 mM Na2HPO4, 2mM KH2PO4, 150 mM NaCl, 2.5 mM KCl, pH 7.5) at a flow rate of 1 ml/minat room temperature. Washes are performed until baseline at 280 nm isreached. GST-BLID and GST-TAT-BLID elution are done by using five columnvolumes of elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH8.0) at a 0.5 ml/min flow rate. The eluted fractions containing theGST-BLID and GST-TAT-BLID recombinant proteins are pooled. Thepurification steps and affinity chromatographic profiles are analyzed byCoomassie Blue-stained SDS-PAGE gels and by western blot analysis usinganti-LEDGF antibody (BD Biosciences).

Afterwards, 20 units of thrombin solution are added per 100 mg of elutedfusion protein and incubated at room temperature for 18 h. Finally, thedigestion reaction mix is loaded on a size-exclusion Sephacryl S-10026/60 High Resolution column (Amersham-Biosciences) equilibrated with200 mM NaCl, 50 mM Tris-HCl, pH 8.0, and the cleaved BLID and TAT-BLIDpeaks are eluted. The chromatographic profile is evaluated by CoomassieBlue, imidazole-stained SDS-PAGE gels and western blot. The purifiedpolypeptides are stored at 4° C. and at −20° C. until further use.

Example 5

Cell Penetrating Peptides (CPP) also known as Protein TransductionDomain (PTD) can also be used to introduce BLID and derivatives intomammalian cells. CPP-BLID constructs are produced via two differenttechnologies: recombinant techniques and chemical synthesis.

An example of a CPP-BLID (CPP-BLID-22) construct is one that comprisesthe first 19 residues of CPP (de Coupade 2005) and SEQ ID NO: 2.

Purified polypeptides from Example 4 and a synthetic CPP-BLID-22 areused in this assay.

HeLa cells are maintained in DMEM (Dulbecco's modified Eagle's medium)supplemented with 10% (v/v) FCS (fetal calf serum), 2 mM L-glutamine and1 mM sodium pyruvate. Cells are seeded and proteins are added 24 hourslater with cells at 60-80% confluence. About 2×10⁵ cells/ml areincubated at 37° C. in 5% CO2 atmosphere in complete culture medium with2, 10, 25 and 75 μg/ml of a CPP-BLID in the presence of 100 μMchloroquine for 2, 4 and 8 hours.

Afterwards at 1 hour, 4 hours, 12 hours, 24 hours and 48 hours intervalsapoptosis is induced by adding to the cells staurosporine (1 uM, or 250nM) for 1.5, 3, 6, 12 and 24 h. Afterwards, cells are analyzed for cellsurvival and apoptosis as described in Example 3.

In this example the overexpression of BLID reduces or abolishes theapoptotic effect produced by the apoptotic stimuli therefore acting asan anti-apoptotic molecule and promoting cell survival. This effect ofBLID constitutes one of the main avenues for drug development based onthis novel protein-protein interaction domain.

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The invention claimed is:
 1. An isolated polypeptide consisting of theamino acid sequence set forth in any one of SEQ ID NO: 1 through 24, ora fragment thereof, wherein said polypeptide or fragment thereof iscapable of protein-protein interaction with Bcl-2 family proteins. 2.The polypeptide of claim 1, wherein said polypeptide or fragment thereofis capable of functioning as an anti-apoptotic factor.
 3. A chimericpolypeptide consisting of a polypeptide consisting of the amino acidsequence set forth in any one of SEQ ID NO: 1 through 24, or a fragmentthereof, wherein said polypeptide or fragment thereof is capable ofprotein-protein interaction with Bcl-2 family proteins directly linkedto a heterologous polypeptide selected from the group consisting offluorescent proteins, glutathione-S-transferase, maltose bindingprotein, beta-galactosidase, inteins, streptavidin, His-tag, mycepitope, HA-tag, and FLAG.
 4. A chimeric polypeptide consisting of apolypeptide consisting of the amino acid sequence set forth in any oneof SEQ ID NO: 1 through 24, or a fragment thereof, wherein saidpolypeptide or fragment thereof is capable of protein-proteininteraction with Bcl-2 family proteins linked through a heterologouslinker consisting of any one of SEQ ID NO: 29 through 32 to aheterologous polypeptide is selected from the group consisting offluorescent proteins, glutathione-S-transferase, maltose bindingprotein, beta-galactosidase, inteins, streptavidin, His-tag, mycepitope, HA-tag, and FLAG.
 5. A chimeric polypeptide consisting of apolypeptide consisting of the amino acid sequence set forth in any oneof SEQ ID NO: 1 through 24, or a fragment thereof, wherein saidpolypeptide or fragment thereof is capable of protein-proteininteraction with Bcl-2 family proteins directly linked to a heterologouscell penetrating peptide, and optionally to a heterologous polypeptideselected from the group consisting of fluorescent proteins,glutathione-S-transferase, maltose binding protein, beta-galactosidase,inteins, streptavidin, His-tag, myc epitope, HA-tag, and FLAG.
 6. Achimeric polypeptide consisting of a polypeptide consisting of the aminoacid sequence set forth in any one of SEQ ID NO: 1 through 24, or afragment thereof, wherein said polypeptide or fragment thereof iscapable of protein-protein interaction with Bcl-2 family proteinsdirectly linked through a heterologous linker consisting of any one ofSEQ ID NO: 29 through 32 to a heterologous cell penetrating peptide, andwherein the chimeric polypeptide may optionally include a heterologouspolypeptide selected from the group consisting of fluorescent proteins,glutathione-S-transferase, maltose binding protein, beta-galactosidase,inteins, streptavidin, His-tag, myc epitope, HA-tag, and FLAG, saidheterologous polypeptide directly linked to the polypeptide or the cellpenetrating peptide, or directly linked to the polypeptide or the cellpenetrating peptide through the linker consisting of SEQ ID NO: 29through
 32. 7. An isolated nucleic acid molecule encoding thepolypeptide of claim 1, wherein said nucleic acid molecule consists ofat least 18 contiguous nucleotides from SEQ ID NO: 26 or SEQ ID NO: 27or consists of the nucleotides from SEQ ID NO: 26 or SEQ ID NO:
 27. 8.An isolated nucleic acid molecule encoding the polypeptide of claim 1,wherein when the nucleic acid molecule is expressed the polypeptideconsisting of the amino acid sequence set forth in any one of SEQ ID NO:1 through 24, or a fragment thereof, wherein said polypeptide orfragment thereof is capable of protein-protein interaction with Bcl-2family proteins is produced.
 9. An isolated nucleic acid moleculeencoding the chimeric polypeptide of claim 3, 4, 5, or
 6. 10. Anexpression vector comprising the nucleic acid molecule any one of claims7 or 8, wherein when the expression vector is expressed the polypeptideconsisting of the amino acid sequence set forth in any one of SEQ ID NO:1 through 24, or a fragment thereof, wherein said polypeptide orfragment thereof is capable of protein-protein interaction with Bcl-2family proteins is produced.
 11. An expression vector comprising thenucleic acid molecule of claim
 9. 12. The expression vector of claim 10or 11, wherein said expression vector is a viral vector.
 13. A host cellcomprising the expression vector of any one of claims 10, 11, or
 12. 14.A method of making a polypeptide of any one of claims 1 to 5, whereinsaid method comprises culturing the host cell of claim 13 and expressingthe polypeptide.
 15. The method of claim 14, further comprisingpurifying the polypeptide.